The Experts below are selected from a list of 150 Experts worldwide ranked by ideXlab platform
Patrick Vinay - One of the best experts on this subject based on the ideXlab platform.
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Proton gradient formation in early endosomes from proximal tubules
Biochimica et biophysica acta, 1996Co-Authors: Vladimir Marshansky, Patrick VinayAbstract:Heavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange. In presence of chloride ions and ATP, the formation of a proton gradient (delta pH) was observed. This process is due to the activity of an electrogenic V-type ATPase present in the endosomal membrane since specific inhibitors bafilomycin and folimycin effectively prevented or eliminated endosomal acidification. In presence of chloride ions (K(m) = 30 mM) the formation of the proton gradient was optimal. Inhibitors of chloride channel activity such as DIDS and NPPB reduced acidification. The presence of sodium ions stimulated the dissipation of the proton gradient. This effect of sodium was abolished by Amiloride Derivative (MIA) but only when loaded into endosomes, indicating the presence of a physiologically oriented Na+/H(+)-exchanger in the endosomal membrane. Monensin restored the gradient dissipation. Thus three proteins (V-type ATPase, Cl(-)-channel, Na+/H(+)-exchanger) present in early endosomes isolated from proximal tubules may regulate the formation, maintenance and dissipation of the proton gradient.
Cornelis A M De Haan - One of the best experts on this subject based on the ideXlab platform.
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dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway
PLOS Pathogens, 2011Co-Authors: Erik De Vries, Donna M Tscherne, Marleen J Wienholts, Viviana Cobosjimenez, Florine E M Scholte, Peter J M Rottier, Adolfo Garciasastre, Cornelis A M De HaanAbstract:Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the Amiloride Derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis.
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Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway
2011Co-Authors: Erik De Vries, Donna M Tscherne, Marleen J Wienholts, Peter J M Rottier, Viviana Cobos-jiménez, Florine Scholte, Adolfo García-sastre, Cornelis A M De HaanAbstract:Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and-independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the Amiloride Derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamindependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all th
Kermit L. Carraway - One of the best experts on this subject based on the ideXlab platform.
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Hexamethylene Amiloride engages a novel reactive oxygen species- and lysosome-dependent programmed necrotic mechanism to selectively target breast cancer cells.
Cancer letters, 2016Co-Authors: Ashley R. Rowson-hodel, Leonardo J. Leon, Anastasia L. Berg, Jessica H. Wald, Jason Hatakeyama, Kacey Vandervorst, Daniel A. Curiel, Colleen A Sweeney, Kermit L. CarrawayAbstract:Anticancer chemotherapeutics often rely on induction of apoptosis in rapidly dividing cells. While these treatment strategies are generally effective in debulking the primary tumor, post-therapeutic recurrence and metastasis are pervasive concerns with potentially devastating consequences. We demonstrate that the Amiloride Derivative 5-(N,N-hexamethylene) Amiloride (HMA) harbors cytotoxic properties particularly attractive for a novel class of therapeutic agent. HMA is potently and specifically cytotoxic toward breast cancer cells, with remarkable selectivity for transformed cells relative to non-transformed or primary cells. Nonetheless, HMA is similarly cytotoxic to breast cancer cells irrespective of their molecular profile, proliferative status, or species of origin, suggesting that it engages a cell death mechanism common to all breast tumor subtypes. We observed that HMA induces a novel form of caspase- and autophagy-independent programmed necrosis relying on the orchestration of mitochondrial and lysosomal pro-death mechanisms, where its cytotoxicity was attenuated with ROS-scavengers or lysosomal cathepsin inhibition. Overall, our findings suggest HMA may efficiently target the heterogeneous populations of cancer cells known to reside within a single breast tumor by induction of a ROS- and lysosome-mediated form of programmed necrosis.
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A cell-permeant Amiloride Derivative induces caspase-independent, AIF-mediated programmed necrotic death of breast cancer cells.
PloS one, 2013Co-Authors: Leonardo J. Leon, Fredric A Gorin, Nagarekha Pasupuleti, Kermit L. CarrawayAbstract:Amiloride is a potassium-sparing diuretic that has been used as an anti-kaliuretic for the chronic management of hypertension and heart failure. Several studies have identified a potential anti-cancer role for Amiloride, however the mechanisms underlying its anti-tumor effects remain to be fully delineated. Our group previously demonstrated that Amiloride triggers caspase-independent cytotoxic cell death in human glioblastoma cell lines but not in primary astrocytes. To delineate the cellular mechanisms underlying Amiloride’s anti-cancer cytotoxicity, cell permeant and cell impermeant Derivatives of Amiloride were synthesized that exhibit markedly different potencies in cancer cell death assays. Here we compare the cytotoxicities of 5-benzylglycinyl Amiloride (UCD38B) and its free acid 5-glycinyl Amiloride (UCD74A) toward human breast cancer cells. UCD74A exhibits poor cell permeability and has very little cytotoxic activity, while UCD38B is cell permeant and induces the caspase-independent death of proliferating and non-proliferating breast cancer cells. UCD38B treatment of human breast cancer cells promotes autophagy reflected in LC3 conversion, and induces the dramatic swelling of the endoplasmic reticulum, however these events do not appear to be the cause of cell death. Surprisingly, UCD38B but not UCD74A induces efficient AIF translocation from the mitochondria to the nucleus, and AIF function is necessary for the efficient induction of cancer cell death. Our observations indicate that UCD38B induces programmed necrosis through AIF translocation, and suggest that its cytosolic accessibility may facilitate drug action.
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Abstract 5489: An Amiloride Derivative capable of inducing ER stress and cell death in highly invasive breast cancer
Experimental and Molecular Therapeutics, 2011Co-Authors: Leonardo J. Leon, William Harley, Kermit L. Carraway, Fredric A GorinAbstract:Metastasis, which is the spread of tumors from their place of origin to other locations in the body via the lymph or blood, is associated with a significant increase in morbidity and mortality. Studies on Amiloride, an FDA-approved diuretic, have shown it to suppress metastatic characteristics such as cell invasion and suppression, and impair their proliferation and viability. Here we show that 38B, a cell-permeant Amiloride Derivative, is more efficacious at causing cell death of cancer cells than Amiloride. We also show that the cell death produced by 38B does not seem to be caused by apoptosis, autophagy, or calcium-dependent necrosis. However, bright field and immunofluorescence microscopy reveal vacuole formation and ER stress in cells, characteristics typically associated with a recently discovered type of cell death known as paraptosis. Since cancer cells have shown to be efficient at evading common forms of cell death such as apoptosis, the discovery of alternative pathways of cell death and a good understanding of the drugs that activate them can be can be key in the improvement of cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2011-5489
Vladimir Marshansky - One of the best experts on this subject based on the ideXlab platform.
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Proton gradient formation in early endosomes from proximal tubules
Biochimica et biophysica acta, 1996Co-Authors: Vladimir Marshansky, Patrick VinayAbstract:Heavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange. In presence of chloride ions and ATP, the formation of a proton gradient (delta pH) was observed. This process is due to the activity of an electrogenic V-type ATPase present in the endosomal membrane since specific inhibitors bafilomycin and folimycin effectively prevented or eliminated endosomal acidification. In presence of chloride ions (K(m) = 30 mM) the formation of the proton gradient was optimal. Inhibitors of chloride channel activity such as DIDS and NPPB reduced acidification. The presence of sodium ions stimulated the dissipation of the proton gradient. This effect of sodium was abolished by Amiloride Derivative (MIA) but only when loaded into endosomes, indicating the presence of a physiologically oriented Na+/H(+)-exchanger in the endosomal membrane. Monensin restored the gradient dissipation. Thus three proteins (V-type ATPase, Cl(-)-channel, Na+/H(+)-exchanger) present in early endosomes isolated from proximal tubules may regulate the formation, maintenance and dissipation of the proton gradient.
Erik De Vries - One of the best experts on this subject based on the ideXlab platform.
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dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway
PLOS Pathogens, 2011Co-Authors: Erik De Vries, Donna M Tscherne, Marleen J Wienholts, Viviana Cobosjimenez, Florine E M Scholte, Peter J M Rottier, Adolfo Garciasastre, Cornelis A M De HaanAbstract:Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the Amiloride Derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis.
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Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway
2011Co-Authors: Erik De Vries, Donna M Tscherne, Marleen J Wienholts, Peter J M Rottier, Viviana Cobos-jiménez, Florine Scholte, Adolfo García-sastre, Cornelis A M De HaanAbstract:Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and-independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the Amiloride Derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamindependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all th